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Prolonged bleeding time with defective platelet filopodia formation in the Wistar Furth rat
P E Stenberg1, R J Barrie, T I Pestina
1Department of Pathology, Oregon Health Sciences University, Portland, OR 97201, USA.
Abstract:
Hereditary macrothrombocytopenia is a hallmark of Wistar Furth (WF) rats. In addition, a platelet/megakaryocyte alpha granule defect, similar to that of patients with gray platelet syndrome, is present. Several observations indicate cytoskeletal abnormalities in WF platelets and megakaryocytes, suggesting the potential for functional defects in hemostatic processes requiring cytoskeletal reorganization, such as platelet adhesion and spreading. However, no bleeding abnormality has been noted. Here, we report a prolonged bleeding time (>30 minutes in 10 of 11 rats tested) with defective clot formation in the WF strain. Prolonged bleeding time can result from defects in platelet adhesion, aggregation, or the release reaction. Because aggregation to collagen and adenosine diphosphate were reported to be normal, we determined whether WF rat platelets are defective in their ability to adhere to substrates. Platelet adherence and spreading was evaluated from 30 seconds to 30 minutes on Formvar-coated, carbon-stabilized grids or poly-L-lysine-coated glass coverslips by transmission electron microscopy or immunofluorescence, respectively, and scanning electron microscopy. We classified the adhered platelets according to their pattern of spreading, ie, rounded, rounded or spreading with short filopodia, spindle-shaped, spreading with long filopodia, spreading with lamellipodia, and fully spread. Adherent normal rat platelets displayed all stages of spreading within 30 seconds to 2 minutes, including many spindle-shaped forms, and forms with multiple, long filopodia. In contrast, adhered WF platelets at these early time points rarely developed long filopodia or were spindle shaped. The majority of adherent WF platelets at these early time points were either round, spread with a few short filopodia, or extensively spread with wide lamellipodial skirts. By 15 to 30 minutes, most platelets in both Wistar and WF samples were fully spread. These data show abnormal WF platelet spreading. The paucity of spindle-shaped forms and forms with long filopodia may reflect an inability of WF platelets to undergo the early stages of spreading, or, alternatively, their more rapid than normal progression through these stages. We hypothesize that this failure to spread normally may relate to prolonged bleeding times in vivo and defective clot formation in WF rats.
Insights
Wistar Furth rats exhibit hereditary macrothrombocytopenia and abnormal platelet spreading, leading to prolonged bleeding times and defective clot formation. These findings suggest cytoskeletal defects impact hemostasis in this rat model.
Area of Science:
- Hematology
- Platelet Biology
- Animal Models of Disease
Background:
- Wistar Furth (WF) rats possess hereditary macrothrombocytopenia and alpha granule defects, resembling human gray platelet syndrome.
- Previous observations suggested cytoskeletal abnormalities in WF platelets and megakaryocytes, hinting at potential hemostatic defects.
- Despite these findings, overt bleeding abnormalities were not previously reported in WF rats.
Purpose of the Study:
- To investigate the hemostatic function in WF rats, specifically focusing on platelet adhesion and spreading.
- To determine if WF rat platelets exhibit functional defects in processes requiring cytoskeletal reorganization.
- To correlate observed platelet abnormalities with in vivo bleeding phenotypes.
Main Methods:
- Evaluation of platelet adherence and spreading on various substrates using transmission electron microscopy, immunofluorescence, and scanning electron microscopy.
- Time-course analysis of platelet spreading patterns from 30 seconds to 30 minutes.
- Classification of adhered platelets based on their morphological spreading stages.
- Assessment of bleeding times in WF rats.
Main Results:
- WF rats demonstrated significantly prolonged bleeding times (>30 minutes) and defective clot formation.
- WF platelets showed abnormal spreading patterns, with a paucity of spindle-shaped forms and long filopodia at early time points.
- While normal platelets rapidly adopted various spreading stages, WF platelets exhibited delayed or altered early spreading, though full spreading occurred by 15-30 minutes in both groups.
Conclusions:
- WF rat platelets exhibit abnormal spreading dynamics, potentially due to impaired early cytoskeletal reorganization.
- The observed defects in platelet spreading correlate with prolonged bleeding times and impaired clot formation in WF rats.
- These findings highlight a link between cytoskeletal function, platelet spreading, and hemostasis in the WF rat model.